Luminous bacteria and methods for the isolation, identification and quantitation of toxicants

ABSTRACT

Methods for the isolation and identification of a toxicant in a sample are disclosed. Luminescent biological agents (i.e., bacteria) having sensitivity to a toxicant or an isolatable component in a sample are used to provide visually discernable zones of luminescent inhibition in the presence of a toxicant (or) in the presence of an isolatable sample component as separated by paper or thin layer chromatography. Kits for use in conjunction with the identification of a toxicant in a sample are also described, which include a luminescent biological reagent as the visualizing agent. Particular examples of luminescent agents include photobacterium leoganthi, photobacterium phosphoreum, Vibrio fischeri, Vibrio harveyi a luminescent fungi, a luminescent fish extract, a luminescent dinoflagellate and fluorescent microorganisms, such as Cypridina. Potential toxicants in a liquid sample, a solid sample, or in a gaseous sample may be identified and further chemically characterized using the described methods. The isolation of potential toxicants in a sample through the processing of a sample through a separation phase matrix such as chromatography paper or TLC plate, followed by exposure to luminescent biological agent, provides for a rapid and inexpensive method for identifying pesticides, herbicides and heavy metals in a known or unknown sample.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the field of methods foridentifying toxicants and/or isolated component substances in a sample.The types of samples which may be analyzed include either a solidsample, a liquid sample or a gaseous sample. The present invention alsorelates to the field of biological toxicant identification agents, as aparticularly described luminescent biological reagent, for example theluminescent bacteria, are employed in the claimed isolation,identification and quantitation methods and techniques disclosed herein.The present invention also relates to the field of toxicant detectingkits, as a kit for the identification of toxicants is describedemploying a luminescent biological reagent.

II. Description of the Related Art

When grown in appropriate liquid culture or on semi-solid culture media,suspensions of luminescent bacteria emit a constant level of light forextended periods. Luminescent bacteria are bacteria which emit lightwithout excitation, (i.e., they glow in the dark). The origin of theemission is biochemical, and organisms which demonstrate thischaracteristic are described as exhibiting the phenomenon ofbioluminescence. Most known examples of luminescent bacteria are marine.Two major subclasses of the luminescent organisms are 1) free living(Vibrio harveyi) and 2) symbiotic (Vibrio fischeri, Photobacteriumphosphoreum, Photobacterium leiognathi). Other major bioluminescentorganisms include fire flies (Photinus pyralis), crustaceans (Cyridinahilgendorfi), dinoflagellates (Gonyaulax polyhedra, Notiluca militaris),fungi (Omphalia flavida) and the sea pansy (Renilla reniformis).

The luminescence of bacteria has long been known to be sensitive to awide variety of toxic substances (e.g., heavy metals, pesticides, etc.).The exquisite sensitivity of luminescent bacteria to a variety ofsubstances has made them a popular choice in methods for the grossdetection of the presence of toxic materials. For example, the use ofluminescent bacteria has been discussed for the detection of toxins onsolid surfaces, such as soil⁵, and in liquid substances, such as in theanalysis of waste water³, as well an in the detection of toxins ingaseous samples⁶.

Luminescent bacteria have also been employed in the detection oftoxicants in marine environments.² For example, Vasseur et al. describea Microtox luminescent bacterial assay for the detection of toxicants inwater (Photobacterium phosphoreum)².

Another variety of luminescent bacteria used in the analysis ofindustrial waste water is described in the Baher patent.³ Specifically,the Klebsiella planticola bacteria has been used to detect the presenceof substances toxic to particular microorganisms (used to purifyindustrial chemical plant waste waters) indicated through monitoring theluminescence of the Klebsiella.

Luminescent bacteria have also been used for detecting the presence ofspecific substances in a sample, including antibiotics, heavy metals,enzyme inhibitors, pesticides, microbial toxins, volatile hydrocarbons,disinfectants, and preservatives.⁶ For example, the Siemens patentdescribes the use of a luciferase-gene-transformed microorganism fordetecting the presence of a toxicant in a sample through a demonstratedreduction in the luminescent signal emitted by the luminescent bacteriain the presence of a toxic substance⁶.

Others have reported the ability to detect the presence of particularclasses of chemical toxicants using luminescent bacteria, particularlyphenolic compounds.⁷ For example, in Strom et al., the relative toxicityof a variety of particularly defined phenolic compounds, includinghydroquinone, is described using a luminescent bacterium⁷.

Thus, some species and components of luminescent bacteria have beenadapted for use to simply detect the general presence of a toxicsubstance in a sample. In the presence of toxicants, detection of thetoxins is provided by an observed diminution in luminescent emission andintensity in a variety of luminescent bacteria. However, the value ofthe "detection" techniques currently available is limited by aninability to identify, in an isolatable form, the substance whichconstitutes the "detected" toxicant or foreign substance.

No methods have been described wherein a generically "detected" toxicantmay be identified in an isolatable form using a luminescent bacteria.The ability to actually identify an isolated substance as a potential"toxicant" in a sample would provide a powerful industrial and researchtool. Moreover, the ability to distinguish, by positive chemicalanalysis, the chemical structure of an isolated toxicant (using variouschemical separation techniques known to those of skill in the art) wouldfind great potential application in research, diagnostic medicine andindustrial manufacturing processes.

Standard chemical visualization techniques for the localization ofseparated substances employ a variety of stains and staining proceduresknown to those skilled in the art (i.e., coomassie brilliant blue forgel electro-phoresis of proteins; 2-Naphthol or Resoranol for paperchromatography of sugars inhydrin for amino acid analysis with TLC).However, these techniques do not identify the potential toxicity of anyvisualized substance in the sample. No system has been proposed whereina reagent may be used to provide a system wherein the potential toxicityof isolated substance in a sample may also be visualized and therebyidentified.

Such a novel method for the simple, inexpensive and sensitiveidentification of a substance(s) in a sample or product which may bepotentially lethal to an organism would also facilitate the furtherchemical elucidation of the chemical identity of the proposed toxicantthrough the subsequent use of various well known chemical analysisstrategies available to those of skill in the art (such as massspectrometry, nuclear resonance spectroscopy, infrared spectroscopy,x-ray crystallography, and chromatographic analysis). Thus, the completechemical structure and identity of the potential toxicant could bedetermined if such a method, capable of identifying in an isolatableform the potential toxicant, were available. Such a system would beparticularly valuable in the development of strategies to remove suchidentified toxicants from products intended for consumer use, and alsoin the development of procedures to render chemically identifiedtoxicant(s) innocuous to animals and humans.

SUMMARY OF THE INVENTION

The present invention provides a rapid and accurate method foridentifying a component substance (such as a toxin/toxicants) in asample through the use of a luminescent biological agent employedtogether with chromatographic resolution techniques.

While any of a variety of luminescent bacteria may be used, thosespecies found to be most particularly preferred for use in the practiceof the present invention include Photobacterium phosphoreum, Vibriofischeri, Vibrio harveyi and Photobacterium leiognathi. However, it isto be understood that the present inventive methods, reagents and kitsmay be practiced using any luminescent organism whose luminescence isspecifically inhibited by an isolated component substance (for example,a potential toxicant) in a sample.

The present methods, reagents and kits may be used to isolate andidentify a single toxicant, a number of individual toxicants, or a groupof toxicants in or on a sample in the solid, liquid, or gaseous phase.

In part, the point of novelty of the present invention resides in theability to identifiably isolate a component substance (for example, atoxicant) contained in a sample rapidly, and without the necessity of aseparate biosensitivity assay of test sample. This is accomplished, forexample, by applying a potentially toxicant-containing sample to aseparation phase matrix, such as a chromatography paper sheet or a thinlayer chromatography plate. The sample-exposed sheet is then exposed toa luminescent biological agent (i.e., the luminescent bacteria)according to the claimed method to accomplish, in one step, both theisolation of each distinct component substance of the sample and thepotential toxicity of each of the distinct components in the testsample.

For example, according to the claimed invention, an unknown sample (forexample a liquid unknown sample or a concentrated extract of a largersample which potentially contains toxicants) may be spotted or streakednear one edge of a chromatography paper sheet at several points.

Most preferably, the sample "spots" or "streaks" are air dried toeliminate the carrier solvent in which the sample was dissolved. Moreapplications of sample(s) can be overlaid onto the respective samplespots, if necessary, and dried. The end of the chromatography sheetclosest to the spotted sample edge is then placed in contact with thesolvent system of choice.

In the usual situation, the solvent of the solvent system will migratethrough the "spotted" sample and through the length of thechromatography paper via capillary action and along the length of thechromatography sheet, thus separating the sample into its componentparts onto particular locations or "segments" on the separation phasematrix (i.e., chromatography paper).

These locations or "segments" of the separation phase matrix (whichprovide the isolated components of the sample) are then exposed to aluminescent biological agent, and provide for the visualiation andidentification of a distinct zone of luminescent inhibition" atlocations or "segments" where luminescent inhibitory components of thesample are located.

Alternatively (to the above paper chromatography method), an unknownsample could be separated using TLC by spotting the sample on a thinlayer chromatography plate. Thus, the sample would be spotted, and airdried analogously to that procedure followed for paper chromatography.However, the solvent in a TLC chamber is at the bottom of the chamberand therefore the solvent migration will be upward through the TLC plateseparation phase matrix.

Depending on a variety of factors, including molecular polarity, theisolatable components in the sample will resolve, on the separationphase matrix, being more soluble in the solvent than having affinity forthe silica gel or other separation phase matrix.

Resolution of the components in the mixture will depend on the polarityof the molecules in the sample verses the polarities of the stationary(e.g. paper, silica or alumina) and mobile (solvent) phases. The endresult in the one dimensional TLC described is a linear array ofcomponents at different locations along the length of the chromatogram.The component substances of the sample thus migrate to isolatablelocations or "segments" on the plate.

Vertical sections along one side or portion of the TLC plate may besprayed with the luminescent biological agent to visualize toxicantlocation. Corresponding unsprayed zones of the plate may then be scrapedoff and eluted with an appropriate solvent or solvent mixture. In thismanner, individual toxicants may be obtained for further separation,chemical identification, or quantitation using those laboratorytechniques well known to those of skill in the art.

More toxicant may be obtained for specific chemical analysis of the thus"identified" locations or segments (areas of luminescent inhibition onthe chromatogram) of the separation phase matrix by eluting identicalsegments from a second run selected separation phase matrix (TLC orchromatography paper) that has not been exposed to the luminescentbiological agent. The chemical structural identity of the toxicant orisolated component substance of the sample may be elucidated accordingto standard laboratory techniques well known to those skilled in theart, such as mass spectroscopy (MS)²² ; high performance liquidchromatography (HPLC)¹⁰,11,12, 28 ; infrared spectroscopy (IR)²³ ;nuclear magnetic resonance (NMR)²²,24 ; thin layer chromatography(TLC)⁹,26 ; x-ray crystallography²²,23 and the like.

As used in the present application, the term "luminescent" biologicalagent is defined as an organism or an extract of an organism, whichemits heatless light under appropriate conditions. Most luminescentsystems involve the use of molecular oxygen. Luciferin (a pigment) and aspecialized form of a luciferase enzyme are included in many luminousorganisms and enables these organisms to emit a heatless light in thepresence of oxygen. Cypridina is an example of a marine organism whichcontains the luciferin pigment. For example, Cypridina contains aluciferin which, when reacted with the Cypridina luciferase enzyme inthe presence of oxygen, emits a heatless bioluminesence. Vibriofischeri¹⁶ and Vibrio harveyi¹⁷ contain an enzyme necessary to makelight, a well as two reagent compounds (a long-chained aliphaticaldehydes and a vitamin derivative, which is a yellow pigment flavinmononucleotide. In reduced form (i.e., in the presence of oxygen) thepigment glows and allows the organism to emit a heatless light. Forexample, Cypridina contains a luciferin which, when reacted with theCypridina luciferase enzyme in the presence of oxygen, emits a heatlessbioluminescence. Similarly, fire flies possess a luciferin pigment whichin the presence of the firefly luciferase and oxygen, provides abioluminescence suitable for use in the practice of the presentinvention. Photobacterium leiognathia is a bacteria which is stronglybioluminescent. All organisms and plants which possess aluciferin/luciferase system would be included among those luminescentbiological agents which could be used in the practice of the claimedinvention.

The present invention also provides a kit for the identification of atoxicant in a sample, which includes a luminescent biological (forexample, bacterial) agent. In a particularly preferred embodiment, thekit comprises a carrier means adapted to receive at least two containermeans and at least one separation phase matrix in close confinementtherewith; at least one separation phase matrix; a first container meanscomprising a luminescent biological agent; and a second container meanscomprising a diluent for the luminescent biological agent.

Most preferably, the luminescent biological agent is a luminescentbacteria, such as Vibrio fischeri (ATCC No. 7744), Photobacteriumphosphoreum, Photobacterium leiognathi, or Vibrio harveyi (ATCC No.33843). In a most preferred embodiment of the kit, the luminescentbiological agent is in a lyophilized form. Where the luminescentbiological agent is in a lyophilized or dried form, the kit will includea diluent suitable for reconstituting the particular biological agentinto its "glowing" form.

By way of example, where the luminescent biological agent is aluminescent bacterial agent, and the particular luminescent bacterialagent is a marine bacteria, a suitable diluent would comprise a saltsolution of at least 1% by weight NaCl. A saline solution between 1% to4% NaCl is even more particularly preferred. Most preferably, thediluent should constitute 3% by weight NaCl.

The diluent of the kit most preferably is a buffering agent whichincludes an NaCl concentration of the diluent should be a concentrationwhich maximizes the luminescent characteristics of the particular marinebacterial species employed. The salt concentration of the diluent hasbeen observed by the Inventors to affect the intensity of the bacteria'sluminescence, and thus the bacteria's suitability as a "visualizing"agent for the described method. For example, where the luminescentbacteria is Vibrio fischeri, a marine luminescent bacteria, the diluentis most preferably about 0.5 M NaCl. Other diluents for marineluminescent bacteria may comprise a saline solution between 0.6-0.66 MNaCl (1%-4% by weight NaCl).

The separation phase matrix may comprise a chromatography paper sheet, aTLC plate, a Sepharose matrix, or virtually any matrix which is capableof separating a mixed sample into discernable, at least partiallyisolated, components. The separation phase matrix most preferred for usein the described kit is a TLC plate.

Most preferably, where the method to be used to isolate the componentsof the sample is paper chromatography, the chromatography paper sheet ismost preferably Whatman chromatography paper 1M or 3M. Where the methodfor separation is TLC, the most preferred TLC plates are Whatmanadsorption plates flexible backed aluminum or polyester #4410-222plates.

The luminescent bacterial agent is to be suspended in a saline solutiondiluen. Where the bacteria is stored in lyophilized form, thelyophilized bacterial agent is reconstituted in the referenced salinediluent to regain its luminescent form prior to use.

Attempts by the Inventors of directly laying a TLC plate on theluminescent bacteria provided relatively low-sensitivity (i.e., a largeamount of inhibitor substance or toxicant needed to be present todemarcate the presence of any isolated substance) for detection, as thediscernable "zones" of luminescent inhibition were relatively faint.Therefore, most preferably, the reconstituted bacterial agent is placedinto an aspirator spray bottle and sprayed onto sample-exposedseparation phase matrix, (for example, the sample-exposed chromatographypaper sheet or TLC plate).

The method of directly spraying a TLC plate with a suspension of theluminescent bacteria was demonstrated to provide the best results, withclearly defined "zones of luminescent inhibition" and wherein even minor(less distinct) zones of luminescent inhibition are discernable. At thistime, spray application of the luminescent biological reagent thusconstitutes the best mode for practicing this aspect of the invention.

However, other methods for achieving contact of the luminescentbiological agent to a test sample may be employed to identify substancesand/or toxicants in a sample. For example, a sheet of film with anagarose or acrylamide layer, or other solid surface or gel containing arehydratable material therein capable of being stored in sheet form andrehydratable prior to use, are contemplated by the Inventors asconstituting equally usable methods for practicing the claimedinvention.

In such an embodiment, a dehydrated form of the luminescent biologicalagent would be incorporated into a porous or water permeable materialwhich was amenable to being formed into a sheet form. The sheet, soimpregnated with a dehydrated form of the luminescent biological agent,would be stored in dry form until needed for use. For use, the sheetwith the bacterial agent in it should be rehydrated in a suitablerehydrating agent, such deionized water or a saline solution. Where theluminescent biological agent is a marine luminescent bacteria, such asVibrio fischeri, the rehydrating agent would most preferably be a salinesolution of at least 1% NaCl. Most preferably, the saline solutionshould be between 1-4% NaCl. A 3% NaCl solution is most preferred.

After the sheet has been rehydrated, the now "glowing" sheet would belaid over a sample of isolated component substances/toxicants to renderthe luminescent biological agent in contact with the test samplecomponent substances. The existance of zones of luminescent inhibitioncould then be examined to identify potential toxicants of the sample.

The claimed invention also comprises a luminescent bacterial agent whichis capable of identifying in isolatable form a component or mixture ofcomponents, substances or a toxicant in a sample. The presence ofisolatable component substances or toxicants in a sample is visualizedthrough the presence of discernable zones of inhibition surrounding theapplied luminescent bacterial reagent (i.e., termed "zones ofluminescent inhibition").

Any luminescent bacteria may be employed in the practice of the presentinvention. However, those luminescent bacterial agents preferred in thepractice of the invention include Photobacterium phosphoreum,Photobacterium leiognathi, Vibrio fischeri, (ATCC Acc. 7744) and Vibrioharveyi (ATCC Acc. 33843). Among these exemplary bacteria, the Vibriofischeri and Vibrio harveyi bacteria embody the even most preferredluminescent bacterial agents of the invention. The Vibrio fischeri (ATCCAcc. No. 7744) constitute the most particularly preferred embodiment ofthe claimed luminescent bacterial agent of the present invention.

As a method for identifying component substances in a sample, using aluminescent biological agent, the claimed method comprises: preparing aluminescent biological agent; obtaining a sufficient volume of thesample to provide a test sample; separating the component substances ofthe test sample by applying the test sample to a separation phase matrixto provide isolated component substances; and exposing the isolatedcomponent substances to a volume of the luminescent biological agent ina concentration sufficient to identify the isolated component substancesof the sample. One or more zones of luminescent inhibition will becomeapparent on the luminescent biological agent-exposed separation phasematrix, and thus identify the isolated component substances in thesample. The concentration of luminescent biological agent sufficient toidentify the isolated component substances of a sample is referred to asa "substance indicating amount". Where the test sample is being analyzedto identify potential toxicant(s), the amount of luminescent biologicalagent is defined as "toxin indicating amount". The necessaryconcentrations to provide this "indicating" effect is between 10⁸ -10⁹bacterial cells/ml of diluent where the bacterial agent is contactedwith the sample in the form of a liquid suspension.

Where paper chromatography is the technique used to separate componentsubstances or toxicants in a test sample, chromatography paper (as theseparation phase matrix) and an appropriate solvent system are used.Corresponding segments on a separate chromatogram (sample pluschromatography sheet) not exposed to luminescent bacteria may be used toobtain additional volumes of the component substances/toxicants of thesample, or where desired, to further chemically identify the isolatedcomponent substances of the sample. Additional sample or chemicalanalysis of the sample in purer form may be accomplished for example, bycutting out the chromatography paper segments (not exposed toluminescent bacteria) which correspond to the identified "zones ofluminescent inhibition"; and eluting the isolated substances from thecut out chromatography paper segments with an appropriate solvent.

The isolated component substances or potential toxicants of the samplemay then be analyzed using standard chemical and spectral means tochemically identify the isolated substances of the sample. If necessary,the eluate of the isolated components of the sample may be concentratedby techniques well known to those skilled in the art prior to chemicaland spectral analysis to chemically identify the isolated substance ortoxicant of the sample.

The luminescent biological agent of the claimed method may comprise aluminescent bacteria, a luminescent fungi, a luminescent fish extract, aluminescent dinoflagellate, a luminescent firefly extract, luminescentanthrogans, luminescent earthworm extract, luminecent coelenterateextract or a luminescent crustacean. (Cypridina organisms).

Most preferably, the luminescent biological agent is a luminescentbacteria, such as Vibrio fischeri (ATCC acc. 7744) Vibrio harveyi (ATCCAcc. 33843), Photobacterium phosphoreum, or Photobacterium leiognathi.The term "luminescent biological agent" as used in the presentapplication may include an organism which has been modified to possessluminescence such as an organism genetically engineered to include theluciferase gene. According to the claimed methods, the test sample maycomprise a liquid sample, a solid sample, or a gaseous sample. Mostpreferably, the sample is to be prepared as a liquid test sample forseparation via a TLC plate separation phase matrix.

While the present methods may be used to isolate and identify virtuallyany substance(s) or toxicant(s) in a sample which is capable ofinhibiting the luminescence of a luminescent biological agent (forexample, a luminescent bacterial agent), preferred applications of thepresent method include the identification of isolated substances such aspesticides, herbicides, heavy metals and their salts, and plantextracts, from a sample. By way of example, pesticides which may beidentified according to the present methods include DIAZANON®, LINDANE®and SEVIN®. By way of example, herbicides which may be identifiedaccording to the present methods include ROUNDUP® and WEED-B-GON®. Heavymetals which may potentially be identified according to the presentmethods include the identification of mercury, lead, cadmium and theirrespective salts.

According to the present method, the isolated substance or toxicant(s)in the sample may be chemically analyzed by any combination oflaboratory techniques well known to those of skill in the art for thechemical characterization of an isolated or partially isolatedsubstance. For example, MS, IR, NMR, HPLC, thin layer chromatography,etc are standard techniques which may be used to further chemicallydefine an isolated substance in a sample. Any of these common laboratorytechniques may be used alone or in combination to identify the chemicalstructure of substantially purified component substances or potentialtoxicants in a sample.

According to one preferred embodiment of the present method, wherein theseparation technique is paper chromatography (separation phase matrix ischromatography paper), the developed chromatogram (having thereupon anyisolatable component substances or toxicants of the sample) may beexposed to the luminescent bacterial agent by spraying a suspension ofthe luminescent bacterial agent, most preferably suspended in a salinesolution, onto the developed chromatogram.

As the agent used to visualize the components/toxicants of a sample isof a biological nature, and therefore potentially sensitive (i.e.,inhibited by chemicals) to components of a desired solvent to be used,failure to remove solvent could in itself cause nonspecific inhibitionof luminescence. Thus, application of the luminescent bacterialsuspension should be done after the complete evaporation of carriersolvent from the chromatogram. In addition, the developed chromatogramshould also be allowed to dry a second time, after the separationsolvent has passed through the sample "streaked" or "spotted"chromatogram, before the luminescent biological (for example,luminescent bacterial agent) is applied (for example sprayed) to thechromatogram.

Observation of a chromatogram exposed to the luminescent agent (the"sprayed" chromatogram) should be made while the chromatogram is stillwet or at least moist with the suspension of luminescent biologicalreagent applied thereto. For example, luminescent bacteria are verysensitive to dehydration, and thus luminescence would be lost everywhereif the investigator does not examine the chromatogram within at least 1hour of exposing the bacteria to the chromatogram. In practice, abacteria-sprayed chromatogram remains moist and glowing from theluminescent biological agent for as long as 45 minutes to one hour,depending on the humidity of the environment.

The Inventors herein demonstrate that the inhibition of luminescence ofparticular species of luminescent bacteria employed according to themethods described herein, is discriminating as among potential toxicantsand/or isolated component substances of a test sample. For example, theInventors have found that the luminescence of one particular species ofluminescent bacteria, Vibrio fischeri, is not inhibited by thepesticide, VOLCK oil spray. Neither does the luminescence of the Vibriofischeri appear to be immediately inhibited by calcium ion. Moreover,all of the luminescent inhibition effects demonstrated through the useof luminescent bacteria, particularly Vibrio fischeri, are concentrationdependent.

The methods of the present invention may be adapted for use in theidentification of closely related components which may be presenttogether in a test sample. For example, selective sensitivities asbetween different luminescent biological agents, particularly as betweenluminescent bacteria, may be used to tailor the disclosed method for usein a particular industry, or to test specific product lines. Forexample, the luminescence of the bacterial agent Vibrio fischeri is moresensitive to the pesticide DIAZANON® than to the pesticide LINDANE®.Similarly, the luminescence of this particular bacterial agent is moresensitive to the inhibitory action of SEVIN® as compared to LINDANE®.Selection of Vibrio fischeri bacteria would thus be indicated asparticularly suitable for use in the described method where a sample issuspected to contain pesticides, such as in a pesticide productionfacility, or perhaps where foodstuffs are stored.

Thus, the particular species of luminescent bacteria may be selected onthe basis of the specific use for which it is intended (i.e., for theidentification of a particular class of related substances). Forexample, where an Investigator wishes to isolate and identify particularpesticides, he/she may select a luminescent bacteria which demonstratesa particular sensitivity to pesticides in general, over another, perhapsless sensitive, luminescent bacteria, for the analysis of a sample whichmay likely include pesticides. Therefore, a hierarchy of relativetoxicant sensitivity, in regard to both the class of toxicant andparticular luminescent bacteria, can be established.

The present invention provides a rapid (about 35 minutes) technique thatcan potentially identify a wide variety of environmentally andbiologically harmful substances.

The Inventors have found that the methods described herein are capableof identifying herbicides and pesticides at their working strengths(i.e., DIAZANON®, LINDANE®, ROUNDUP® AND WEED-B-GON® diluted 1/150).Therefore, herbicides, pesticides and other environmental pollutants andcontaminants may be identified according to the present method with thedescribed kits as they occur in the environment in the air, in lakes,streams, ground water and in run-off from fields, for example, inrelatively dilute form (for example diluted 1/1,000 from commercialstock concentration).

As used in the present disclosure, the term "toxicant" and "identifiedisolated component substance" of a sample is defined as a substancewhich is capable of inhibiting the luminescence of a luminescentbiological agent, such as a luminescent bacteria, Vibrio fischeri.

Even more specifically, the term "toxicant" is broadly defined as asubstance which is capable of inhibiting or potentially lethal to, avirus or a living organism, such as a plant, animal or microorganism.Even more specifically a toxicant potentially toxic to an animal such asa human may be identified using the described method. Toxicity tobacteria is recognized as an indication of toxicity of a substance tohigher organisms, including humans. The Inventors hypothesize that formsof the biological agents which are represented by whole organisms,rather than extracts of whole organisms, will be both more sensitive andalso be capable of identifying a broader range of substances andtoxicants in a sample in smaller concentrations than with luminescentextracts from an organism.

As used in the present application, the term bioluminescence morespecifically refers a living organism or from extracts of a livingorganism when combined under appropriate conditions. Lack ofluminescence refers to the lack of light emission not necessarilyrelated to the expiration of the organism.

The following abbreviations are used throughout the Specification:

ECD=Electron Capture Detection

TLC=Thin Layer Chromatography

NMR=Nuclear Magnetic Resonance Spectroscopy

M=Molar

HPLC=High Performance Liquid Chromatography

IR=Infra-Red Spectroscopy

MS=Mass Spectroscopy

D=Dimension

THF=Tetrahydrofuran

UV=Ultraviolet

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 TLC plate with garlic extract sample in H₂ O. Not exposed toluminescent bacteria. TLC Plate identified those components which areultraviolet light-absorbing. If compound absorbs the 254 nM light, thenarea where compound is located will not glow, and appears as a dark spot(V shape) in a garlic extract using fluorescent detection (254 nMexcitation). Solvent used is 80:8:12 mixture of (H₃ CCN:H₂ O:NH₃). TheV-shaped areas are not indicative of a bacteriotoxic agent. Results fromthese analysis indicate a compatible system for resolving ultravioletabsorbing and thereby identifiable components in a sample.

FIG. 2 TLC plate with garlic extract sample exposed to luminescentbacteria, Vibrio fisheri, same solvent as FIG. 1. Bioluminescenceinhibition is evident as a dark circular region (about 10.5 cm frombottom of plate). This circular region is hypothesized to constituteallicin in the garlic extract.

FIG. 3 TLC plate with DIAZANON® and LINDANE® by fluorescence.

FIG. 4 TLC plate DIAZANON® and LINDANE® by bioluminescence.

FIG. 5 TLC for DIAZANON® dilution series. Plates demonstrate a dilutionseries of DIAZANON®. The presence of DIAZANON® is demonstrated at dimareas defining the "zone of luminescent inhibition" of the luminescentbacteria, Vibrio fischeri, in response to the pesticide. Dilutionsemployed of the pesticides were full strength, 1:128; 1:256; 1:512 and1:1024.

FIG. 6 TLC for DIAZANON® dilution series with the luminescent bacteria,Vibrio fischeri (same dilutions as for FIG. 5).

FIG. 7 TLC of DIAZANON® with either UV 254 fluorescence orbioluminescence inhibition with luminous bacteria, Vibrio fischeri in asample.

FIG. 8 TLC of DIAZANON®, ROUNDUP® and WEED-B-GON® identified at adilution of 1/150 (working strength). Luminescent bacteria exposure timeprior to examining the bacteria-sprayed plates was 35 minutes.

FIG. 9 TLC plates of two pesticides, DIAZANON® and LINDANE® and twoherbicides, ROUNDUP® and WEED-B-GON®, taken in room lighting. Dilutionof pesticides and herbicides=1/150.

FIG. 10 TLC plates of two pesticides, DIAZANON® and LINDANE® and twoherbicides, ROUNDUP® and WEED-B-GONE® viewed by bioluminescence showingzones of luminescent inhibition.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods, kits and luminescent biological(for example, bacterial) agents which are demonstrated to besurprisingly advantageous for the identification of specific toxicantsor component substances in a sample. Moreover, techniques are proposedwherein the identified component substances of a sample may subsequentlybe chemically characterized or additional volumes of the isolatedcomponent ingredient (i.e., toxicant) be obtained employing a variety ofchemical techniques in conjunction with the teachings of the presentdisclosure.

The novel use of a luminescent biological agent together with aseparation phase matrix provides a unique method for the rapid andsimple identification of potentially toxic (isolated) substances in asample. The Inventors foresee the application of the present inventionin the laboratory as well as in industry for the detection ofenvironmental pollutants, particularly in water resources. Additionally,use of the described methods in the development and identification oftherapeutically valuable components in plants and organisms, such as ingarlic, is also considered an important application of the describedinvention.

Luminescent Bacteria as the Luminescent Biological Agent

Where the luminescent biological agent to be used is a luminescentbacterial agent, such as the luminescent bacteria Vibrio fischeri, thebacteria should constitute a suspension of bacteria at a finalconcentration of about 10⁸ -10⁹ bacteria cells/ml in the suspension tobe used, for example, where the luminescent bacterial agent is sprayedonto a chromatogram.

A preferred method whereby the luminescent bacteria are prepared for usein the presently described invention is as follows. The bacteria mustfirst be allowed to become fully "induced" in their luminescent system,i.e., the luminescent system of the bacteria should be allowed to reachcomplete development prior to harvesting of the bacteria from theculture. Determination of at what point a bacteria has reached fullluminescent system development is well known to those of skill in theart³⁰,31.

Upon full development of the luminescent system of the bacteria, thebacteria should be harvested and then placed in a centrifuge tube. Thebacteria are then to be centrifuged at a speed of 10,000×G for 30minutes at room temperature. Thus centrifuged, the bacteria will form apellet of cell "paste" at the bottom of the tube. About 1 gram of thiscell paste (about 12 ml of cell "paste"=1 gram) of glowing bacteria isthen to be diluted to a volume of 20 ml, by adding 20 ml of a diluent ofchoice. Where the luminescent bacteria is a marine bacteria, forexample, the diluent is most preferably a buffered saline solution ofbetween 1-4% NaCl. As diluted to 20 ml, the cell suspension constitutesa concentration of 10¹⁰ -10¹² bacteria cells/20 ml (or 10⁸ -10⁹cells/ml).

The following Examples are presented only to describe preferredembodiments and utilities of the present invention, and to satisfy bestmode requirements. The examples are not meant to limit the scope of thepresent invention unless specifically indicated otherwise in the claimsappended hereto. The following Examples are provided to demonstratevarious aspects of the present invention.

EXAMPLE 1

Isolation of Identifiable Luminescent Inhibitory Toxicant in GarlicExtract Using Luminescent Bacteria

PROPHETIC EXAMPLE 2

Proposed Chemical Identification of Toxicants in a Garlic Extract

EXAMPLE 3

Identification of Pesticides in Sample with Luminescent Bacteria

EXAMPLE 4

Dilution Series of DIAZANON® or TLC with Vibrio fischeri

EXAMPLE 5

Solvent Polarity and Fluorescent and Bioluminescent Detection ofDIAZANON®

EXAMPLE 6

Identification of Pesticides and Herbicides in a Sample with LuminescentBacteria

EXAMPLE 7

Identification of Herbicides and Pesticides

PROPHETIC EXAMPLE 8

Proposed Identification of Heavy Metals in a Sample with LuminescentBacteria

PROPHETIC EXAMPLE 9

Proposed Chemical Identification of a Toxicant in a Sample Isolated withBioluminescence Methods

EXAMPLE 10

Identification of Toxicant in a Gaseous Phase Sample with LuminescentBacteria

PROPHETIC EXAMPLE 11

Proposed Identification of a Toxicant on a Solid Surface Sample withLuminescent Bacteria

PROPHETIC EXAMPLE 12

Proposed Test Kits for Identifying Toxicants in a Sample

EXAMPLE 1 Isolation of Identifiable Luminescent Inhibitory Toxicant InGarlic Extract Using Luminescent Bacteria

The present example is presented to describe a method by whichcomponents of a substance which inhibit luminescent bacteria may beisolated. The sample analyzed in the present example is a garlicextract. For this experiment, the Inventors first prepared a garlicextract from garlic powder. The garlic powder was processed so as toform a liquid garlic extract. One (1) gram of garlic powder was blendedwith 5 ml. H₂ O. Other solvents such as ethanol, chloroform, or acetonemay be used to blend the sample, but H₂ O was found to be the bestsolvent for the garlic.

A 5 ml. volume of the garlic extract was first applied ("spotted") toTLC plates at several points equidistant from one edge of the plate. Theplate was inverted in a sealed TLC solvent container with a small amountof solvent in the bottom such that spotted samples were parallel to andabove the solvent interface.

As the solvent (acetonitrile:water:aqueous ammonia, 8:1.5:0.5) migratedup the TLC plate, the individual components in the garlic extract weresufficiently separated to detect separate zones of luminescentinhibition upon exposing the developed chromatogram to a suspension ofluminescent bacteria, Vibrio fischeri applied in a suspension of 0.5 MNaCl (See FIG. 1 and FIG. 2).

The Inventors applied the luminescent bacteria, Vibrio fischeri to thechromatogram specifically by spraying the described suspension ofbacteria (contained in a buffered salt solution of 3% (0.5 M) NaCl at apH of about 7) onto the developed chromatogram after the solvent inwhich the sample was contained had evaporated. Zones of luminescentinhibition were located prior to the dehydration of the bacteria on thechromatogram, i.e., at least within 1 hour after application of thebacteria.

The inhibition of bioluminescence of the bacteria caused by the presenceof toxicants in isolated components of the garlic extract was thenvisualized. The bioluminescent inhibition effect of any toxicant in thegarlic extract became apparent generally within a few minutes in theform of a clearly demarcated zone of bioluminescent inhibition (See FIG.2). These zones of bioluminescent inhibition are areas on thechromatogram which were dimmer (i.e., less brightly emissive) than themore brightly emissive surrounding areas on the chromatogram (which didnot include isolated components of the garlic extract which were capableof inhibiting the luminescence of the Vibrio fischeri). The areaswherein the chromatogram demonstrated greatest amounts and intensity ofblue bioluminescence from the applied Vibrio fischeri bacteriaidentified areas of no component substances or instead isolatedcomponents of the garlic extract which were not toxic to thebioluminescence of the bacteria, and therefore according to thedescribed method were considered not to constitute toxicants.

As stated, the inhibition of bacterial luminescence which occurs when atoxicant is detected, becomes apparent very soon, often within a fewminutes, and grows more distinct with time and reaching a pronouncedpeak effect in the minutes before the chromatogram dries out, i.e., thezones of decreased luminescence show more contrast relative to thesurrounding luminescence with time, prior to the chromatogram dryingout. When the chromatogram is dried out, of course, all the luminescenceof the bacteria on the chromatogram will be extinguished with thedehydration of the bacteria.

Curiously, with the described methods, those positions on thechromatogram to which the toxicants have migrated (i.e., the "zones ofinhibition") appear to dry out faster than the remainder of thechromatogram which, for example, remains highly luminescent.

Alternatively, the identification of the different individual componentsof the garlic extract could have been accomplished using paperchromatography as the separation phase matrix for the sample or othersuch techniques well known to those of skill in the art.

FIG. 1--TLC Plate of Garlic Extract

This is a photograph of a TLC plate viewed by fluorescence. The actualplate was 20 cm by about 4.5 cm. In the photograph the plate is seenreduced to 13.0 cm by 2.95 cm. Dimensions below refer to dimensions ofthe photograph not of the original plate.

Sample

Aqueous Garlic Extract. Preparation: 1.0 g of powdered garlic suspendedin 5.0 ml of H₂ O. Mixed with Vortex mixer for 1 minute. Centrifuged intable top centrifuge on high (about 1-2000 rpm, 60 seconds) to obtainstraw yellow supernatant: the sample. Five μl applied at origin ofplate: pencil lines seen near 1.7 cm from bottom of plate. Theapplication zone is seen as a circle (faint) of about a 6 mm diametercentered on the line.

Development

The solvent system used was acetonitrile:water:25% ammonia (aqueous);80:8:12. The sample was chromatographed in a closed chamber forapproximately 20 minutes. The solvent front traveled about 4/5 of thedistance of the plate. A faint demarkation line is seen at about 10.5 cmfrom the bottom of the plate showing the location of this solvent front.

Results

Major features of the chromatogram viewed by fluorescence excitation area pronounced dark line at about 7.9 cm from the bottom of the plateseveral chevron or V-shaped dark areas in the 5.8-7.7 cm from the bottomregion and a faint roughly circular shaped zone centered at about 8.8 cmfrom the bottom. The chevron shaped darkenings represent chemicalcomponents in the garlic resolved by the chromatographic process. Themore or less circular zone at 8.8 cm (which can be more dramaticallyrevealed by moving the photograph back and forth about 2 cm in the planeof the photograph) is the zone or near the zone of bioluminescenceinhibition seen in photograph 2.

FIG. 2--TLC Plate of Garlic Extract

This is a photograph of a TLC plate (not the same one as in photograph1, but a plate developed in an identical fashion except for a longertime) viewed by the emission of Vibrio fischeri luminous bacteria. Theactual plate was 20 cm by about 4.2 cm. In the photograph the platedimensions are 12.9 cm by 2.8 cm.

Sample

Aqueous garlic extract: the identical sample used in chromatogram ofphoto 1.

Development

Same as in photo 1 except chromatogram ran longer, front reaching nearthe end of plate, near 12.5 cm in photograph. The origin was centered onthe pencil line visible at about 2.4 to 2.5 cm from bottom of plate.

Results

A very dark, nearly circular zone is seen centered at about 10.5 cm frombottom of plate. A faint second zone is seen at about 6.7 cm from thebottom. Several darkened regions can be seen at the edges of the plate.The dark areas which appear at the edges are artifacts, and representplaces on the chromatogram sheet which were not adequately sprayed withthe luminous bacterial suspension. The zone at 10.5 cm represents thelumotox effect i.e., the determination of the location of the componentin garlic which inhibits the luminous bacteria.

Routinely, for preliminary analysis of the chromatograms, the plateswere irradiated with a lamp emitting UV (254 nm) radiation. The TLCplate used had the F₂₅₄ backing and were therefore fluorescenteverywhere that no UV absorbing samples or components existed. Thispreliminary detection system also revealed component substances as darkspots on a light background where heterocyclic or other UV absorbingcompounds were present. However, fluorescent extinction and luminescenceinhibition were often not in parallel. For example, some samplespresented as very dark zones, as viewed by fluorescence (for example,garlic), had little or no bioluminescence inhibition, while other zonespresented very faint or non-existent fluorescence extinction but hadsubstantial ability to inhibit (extinguish) bioluminescence (e.g.,garlic, LINDANE®, ROUNDUP®).

Particular sources of TLC plates and chromatography sheets includeSargent Welch (No. S18953-10-TLC plate with F₂₅₄ fluorescent material),Analtech (uniplate taperplate silica gel G-F, No. 81013), andEastman-Kodak (Kodak chromatogram sheets silica gel absorbent withfluorescent indicator, catalog no. 122-4294) and Whatman (absorbentplates flexible-backed aluminum polyester, catalog no. 4410-22 (containsfluorescent indicator)).

The Albert et al. article²² provides a description of analyzingmevinolin, a fungal metabolite employing standard laboratory techniquessuch as mass spectroscopy, nuclear magnetic resonance and x-rayanalysis. These alternative standard laboratory techniques could beutilized to analyze eluted components from an unknown sample.

Upon isolation/separation of the various components in the garlicextract sample by a chromatography method, the inventors then appliedthe luminescent bacteria to the developed chromatogram. Most preferably,the luminescent bacteria is applied to the developed chromatogram in theform of a suspension contained in a buffered salt solution (about 0.3 MNa⁺ /K⁺ phosphate buffered saline (3% NaCl by weight) pH 7.0).

Prophetic Example 2--Proposed Chemical Identification of Toxicants in aGarlic Extract

The present prophetic example is provided to outline one proposed methodby which the toxicant(s), as identified according to the method of theprocedure outlined in Example 1 may be further characterized to identifythe chemical structure of the isolated toxicant(s). This method may alsobe used where additional amounts of the isolated substance are desiredor where the purity of the isolated substance is to be determined.

The particular "zones of luminescent inhibition" described above, whichprovide for the isolation of the component substances (i.e., toxicant)in the test sample, are used as reference points to isolate eachcomponent substance from an adjacent spotted sample which was run on thesame or a separate TLC plate with the same sample. Unsprayed sections ofthe TLC plate, which correspond to zones of luminescent inhibition onthe sprayed portion, may be scraped off and added to a sufficient volumeof an appropriate solvent (i.e., distilled water, acetone, ethanol,ether, ethyl acetate-chloroform or other solvent mixtures) such that theisolated component substance of the sample may become dissolved in thesolvent.

Subsequent removal of the solid TLC scrapings from the liquid eluate canbe accomplished by various methods known in the art such ascentrifugation or filtration. If necessary, the eluates containingdissolved toxicants may then be concentrated using standard techniques.These separated, (and in some cases, concentrated) isolated substancesof the sample may be further resolved in other TLC solvent systems (orHPLC, paper chromatography, and the like) to verify purity or to obtainsuitably pure isolated substances. These substantially pure isolatedsubstances can then be identified using standard chemical and spectralmethodologies. For example, such standard chemical and spectralmethodologies include as HPLC, MS, IR, NMR, and the like.

Alternatively, two dimensional (2D) thin layer (TLC) can be run forhigher resolution of the sample for more explicit identification ofcomponents therein. In the 2D method, a sample is spotted near onecorner of the TLC sheet or plate, and run successively in two, usuallyperpendicular, directions, using different solvent systems orconditions. For example, the sample is chromatographed in the usual way(described above) on the TLC medium in the first direction using solventsystem No. 1 (e.g., a basic non-polar system, ammonia:butanol:hexane ina 5:20:75 ratio). The chromatogram, containing components resolved in alinear fashion in this solvent system No. 1, is then to be removed fromthe chromatography chamber, dried fully to remove solvent molecules ofthis system No. 1 solvent, and then the thus dried chromatogram isrotated 90° to the orientation first used and chromatographed in the neworientation using a solvent system No. 2 (e.g., a polar, acetic system,such as acetic acid, acetone, ethanol in a 10:50:40 ratio). Thecomponents resolved into a linear array by system No. 1 move in theperpendicular direction with the solvent system 2 to provide evengreater resolution of individual component substances in the sample.

This same basic approach can be utilized where luminescent bacteria areused to identify isolated component substances of a sample separated bypaper chromatography systems, either 1D or 2D. As those in the art willappreciate, in using such systems, there are various ways to achieveseparation such that toxicants can be obtained in relatively pure form.For example, another version of 2D paper chromatography may employelectrophoresis in one dimension and gravitational flow paperchromatography or isoelectric focussing in another dimension, or othertwo-dimension combination thereof (i.e., 1st D=paper chromatography, 2snD=isoelectric focusing, etc.)

Example 3 Identification of Pesticides in Sample with LuminescentBacteria

The present example is provided to demonstrate the use of the claimedmethods and reagents for the identification of a pesticide in a sampleof known substances. In this example, the pesticides identified wereDIAZANON®, LINDANE® and SEVIN®. The luminescent bacteria used in thepresent example was Vibrio fischeri (ATCC 7744).

Identification of these individual pesticides and herbicides wasachieved essentially according to the same methods described inExample 1. A suspension of Vibrio fischeri in a saline diluent wassprayed, using an aspirator bottle, on the developed chromatograms.Zones of luminescent inhibition appeared surrounding those areas on theplate where the DIAZANON® had migrated. Similar, less dim zones ofinhibition, where LINDANE® had migrated (See FIG. 6 and FIG. 7). In asimilarly run TLC with SEVIN®, the chromatogram also demonstrated zonesof luminescent inhibition at those areas on the chromatogram whereSEVIN® had migrated.

FIG. 3 and FIG. 4

These are photographs of the same TLC plate taken by two differentconditions: Fluorescence and Bioluminescence, respectively.

Samples

5 μl samples of (1/32 by DIAZANON®) and (1/8 LINDANE®). The DIAZANON®sample was produced by serial dilution of the commercial diagram (25%w/v) 0,0,diethyl-0-[2-isopropyl-6-methyl-≮-pyrimidinyl]phosphorsthionate, OrthoProducts. The DIAZANON® was diluted with ethanol by factors of 2 until adilution of 1/32 commercial strength was reached. The LINDANE® (OrthoProducts) was diluted in ethanol from the commercial 20% (w/v) gammaisomer of benzene hexachloride, until a final strength of 1/8 wasreached.

Development

Acetonitrile: 25% Aqueous ammonia, 75:25

Results

FIG. 3 represents the results from this study using DIAZANON® andLINDANE® on a TLC plate viewed by 254 nm excitation. A prominent darkzone for DIAZANON® is located at 8.8 cm from bottom of FIG. 3. About 3quite faint zones for LINDANE® at 8.2, 9.2, and 10.3 cm from bottom ofFIG. 3 are demonstrated. DIAZANON® origin (application spot) at 2.5 cmfrom bottom of photo, LINDANE® origin at 3.0 cm.

FIG. 4 viewed by bioluminescence from Vibrio fischeri. Dark zone forDIAZANON® very close to zone for fluorescence extinction (at about 8.1cm from photobottom). Several very dark zones for LINDANE® at about 8.0,8.8, and 10.0 from photobottom. Also seen is slight inhibition zone atorigin of LINDANE® sample. The several zones for LINDANE® indicate thatseveral isomers or different inhibition compounds are present in theLINDANE® sample.

Example 4 Dilution Series of DIAZANON® on TLC with Vibrio fischeri

The present example is provided to demonstrate the sensitivity of theclaimed invention to detect relatively low concentrations of apesticide. An exemplary pesticide for demonstrating the sensitivity ofthe assay used here is DIAZANON®.

FIG. 5 and FIG. 6

Spot tests of DIAZANON® at several dilutions were performed at thefollowing strengths: full strength (25% w/v DIAZANON®), 1:128; 1:256;1:512; and 1:1024. No chromatography was done. 5 μl samples of thevarious DIAZANON® dilutions were applied to TLC plate material, sprayedwith a suspension of Vibrio fischeri in a saline solution (3% NaClWT/VOL.) and photographed. Marked inhibition occurred up to andincluding the D/256 dilution (D/252 appears by clerical mistake on sheetinstead of D/256 which was used) of full strength (25% w/v) DIAZANON®.Faint inhibition is seen at dilution 1:512 and dilution 1:1024 (See FIG.6, R).

The TLC plates with DIAZANON® demonstrate that the methods describedherein are sufficiently sensitive to identify a pesticide in a sample atconcentrations in which they are likely to occur in a land or watersample obtained in the environment.

Example 5 Solvent Polarity and Fluorescent and Bioluminescent Detectionof DIAZANON®

The present example is presented to demonstrate the effect of varyingthe solvent polarity on the detection patterns, or "zones of inhibition"of Vibrio fischeri in the presence of DIAZANON®, a pesticide.

FIG. 7 and FIG. 8 provide photographs of TLC plates viewed by 254 nmirradiation (FIG. 7) and by bioluminescence (FIG. 8).

Samples

In each case, 5 μl of (DIAZANON®/8) was applied at origin on left and 5μl of LINDANE®/8 was applied at right origin.

Development

Three solvent systems used. All composed of Hexane: THF mixtures. InFIG. 7 the left chromatogram was Hex:THF, 70:30 the middle chromatogramwas Hex:THF, 80:20 the right chromatogram was Hex:THF, 90:10. (middlechromatogram contains clerical labeling error of 80 THF:20 HEX, whichshould be 80 HEX:20THF)

Results

FIG. 7 shows the decrease in polarity as the proportion of THFs loweredcauses the DIAZANON® and faint LINDANE® spots or zones to beprogressively diminished in mobility; to have smaller R_(f) values; tomigrate shorter distances from the origin.

FIG. 8 shows only the left-hand and right-hand TLC plates seen in FIG.9. Dark bioluminescence zones of inhibition are seen in Photo 8 forDIAZANON® and LINDANE® samples.

Example 6 Identification of Pesticides and Herbicides in a Sample withLuminescent Bacteria

The present example is provided to demonstrate the use of the claimedmethods and reagents for the identification of pesticides and herbicidesin a known test sample using a luminescent biological agent.

In this example, the herbicides ROUNDUP® and WEED-B-GON® and thepesticides DIAZANON® and LINDANE® are identified in a test sample withthe luminescent bacteria, Vibrio fischeri (ATCC 7744).

Example 7 Identification of Herbicides and Pesticides

Each sample was run on an individual TLC sheet. Photographs of theresulting 4 individual chromatograms are presented at FIG. 9 (roomlight) and FIG. 10 (Bioluminescence--chromatogram with luminescentbacteria).

Two solvent systems were used. The solvent systems used to identify theherbicides (ROUNDUP® and WEED-B-GONE®) was 100% ethanol. A 5 ml sampleof an 8-fold dilution of these commercially available herbicides wasused in the spotting of the TLC plates.

The solvent system used for the pesticides DIAZANON® and LINDANE® wasHexane:THF, 90:10. The pesticides were spotted at a concentration of1.8. A 5 ml sample of an 8-fold dilution of these commercially availablepesticides was used in the spotting of the TLC plates.

Two solvent systems were employed as no single system has yet been foundto adequately resolve all compounds (i.e., the two pesticides and thetwo herbicides). Use of 100% ethanol causes DIAZANON® and LINDANE® torun at the front of the solvent system. Use of 90% Hexane, 10% THFcauses ROUNDUP® and WEED-B-GONE® to stay at the origin. The TLC platesphotographed are in the following order (left to right) (one sample perplate): DIAZANON®, LINDANE®, ROUNDUP®, and WEED-B-GONE®. In each case,the commercial strength was diluted by a factor of 8.

Results

a. Pesticides

The DIAZANON® sheet did present an entirely distinct "zone ofinhibition", but the FIG. 10 only marginally indicates thischaracteristic, perhaps due to partial bacteria dehydration. TheLINDANE® chromatogram presented as a distinct zone of inhibitionculminating in a dark spot center about 2.9 cm from the bottom of theplate.

The ROUNDUP® chromatogram presented a clear zone of inhibition as seenat the origin, and at least one other inhibition zone centered at 4.5 cmfrom the bottom of the plate. The WEED-B-GONE® chromatogram presented asa large oval zone of luminescent inhibition (perhaps comprised ofseveral components) starting at about 1.8 cm from the bottom of the TLCplate and stretching to beyond 6 cm from the bottom of the plate.

Example 7 Identification of Herbicides and Pesticides

The following example presents the results of three separately runexperiments by the Inventors. These data demonstrate the reliability ofthe described methods for consistently identifying a component substancein a sample. The following list represents a description of theparticular herbicides and pesticides, and the percent dilutions usedthereof, in the described 3 separately run TLC plates.

WEED-B-GON®

10.8% w/v dimethylamino salt of 2,4 dichlorophenoxyacetic acid 11.6% w/vdimethylamino salt of 2-(2-methyl-4 chlorophenoxy)propionic acid

DIAZANON®

25% w/vO,O,diethyl-O-[2-isopropyl-6-methyl-4-pyrmidinyl]phosphorothio(n)ate

ROUNDUP®

41% w/v isopropylamino salt of glycophosphate N-(phosphoromethyl)glycine

LINDANE® (bark and leaf mineral spray)

20% w/v gamma isomer of benzene hexachloride liquid

LIQUID SEVIN® CARBAMYL

27% w/v 1-naphthyl-N-methyl carbamate

The following table presents the results obtained for identifyingDIAZANON®, LINDANE®, ROUNDUP®, and WEED-B-GON® in three different testsconducted by the Inventors. These data demonstrate that the describedmethod provides a system which possesses the ability to detect, withvarying sensitivity, a variety of herbicides or pesticides in a sampleon a consistent and reliable basis, as demonstrated by the closelycorresponding "spots" for each run of the same component substancebetween the three separately run chromatograms.

                  TABLE 1                                                         ______________________________________                                                                            Ratio of                                    Herbicide/ Distance Spot Spot Spot Front Stan.                              Pesticide of Front 1      2    3    Fluor                                                                              Biolu                                                                              Dev.                            ______________________________________                                        Test 1                                                                          DIAZANON ®  2.38 0.63 -- -- 0.26 0.26 0.02                                LINDANE ® 2.38 .88 1.38 1.66 0.37 0.37 0.02                                    0.58 0.57 0.00                                                                0.70  --                                                                 ROUNDUP ® 2.36 0.00 0.13 1.38 0.00 0.00 0.00                                   0.06 -- --                                                                    0.58 -- --                                                               WEED-B- 2.36 0.66 1.64 1.88 0.28 0.28 0.02                                    GON ®     0.69 0.69 0.04                                                       0.80 -- --                                                               Test 2                                                                        DIAZANON ® 2.75 0.68 -- -- 0.26 0.25 0.01                                 LINDANE ® 2.19 0.83 1.23 1.55 0.38 0.35 0.03                                   0.56 0.57 0.00                                                                0.70 -- --                                                               ROUNDUP ® 2.00 0.00 0.12 1.22 0.00 0.00 0.00                                   0.06 -- --                                                                    0.61 -- --                                                               WEED-B- 2.25 0.65 1.35 1.79 0.28 0.28 0.020                                   GON ®     0.66 0.66 .01                                                        0.80 -- --                                                               Test 3                                                                        DIAZANON ® 2.79 0.68 -- -- 0.24 0.24 0.00                                 LINDANE ® 2.09 0.83 1.23 1.59 0.39 0.40 0.01                                   0.58 0.60 0.03                                                                0.73 -- --                                                               ROUNDUP ® 2.13 0.00 0.21 1.29 0.00 0.00 0.00                                   0.10 -- --                                                                    0.60 -- --                                                               WEED-B- 1.84 0.38 1.17 1.55 0.21 0.21 0.05                                    GON ®     0.64 0.63 0.02                                                       0.84 -- --                                                             ______________________________________                                         R.sub.f Values Represented in the Reported Values in the Table;               R.sub.f = relative to the front; a fractin of the total distance which th     solvent front migrated.                                                  

Prophetic Example 8 Proposed Identification of Heavy Metal Salts in aSample with Luminescent Bacteria

The present prophetic example is provided to present a use of theclaimed methods and reagents for the identification of a heavy metal ina sample. Specifically, the Inventors hypothesize that the describedmethods would be useful in the identification of the heavy metals suchas mercury, lead and cadmium using the described luminescent biologicalreagents, such as the bacteria, Vibrio fischeri (ATTC Acc. No. 7744).

In the present example, the Inventors spotted the various metals on to achromatography paper sheet, but did not run them through achromatography separation process. Upon spotting of the various metalsalong one side of a chromatography paper sheet, the sample spots wereallowed to dry. Upon drying, the spotted sheets were exposed to theluminescent bacteria Vibrio fischeri. Employing this method, theInventors were able to visualize the presence of the heavy metal saltsof mercury, lead, and cadmium.

To isolate the heavy metal spotted on the chromatography paper, thepaper edge at which the sample was spotted should be exposed to asolvent system, most preferably an acidic solvent system.

Specific reference is made here to the RAININ® catalog²⁹, wherein astandard technique (for the separation of heavy metals) is describedusing an ion chromatography metals column. Resolution of Pb⁺⁺ and Cd⁺⁺is demonstrated in the reference RAININ® catalog.

Successive equal volumes of a heavy metal could be eluted using the HPLCprocedure from the HPLC machine and spotted in an array or in a linearfashion on a sheet of (Whatman) chromatography paper. After the carriersolvent is evaporated or otherwise removed by drying, the sheet could besprayed with a suspension of luminescent bacteria, such as Vibriofischeri, as described. Zones of bioluminescent inhibition could besimilarly visualized to identify the metal.

Prophetic Example 9--Proposed Chemical Identification of a Toxicant in aSample Isolated with Bioluminescence Methods

The present prophetic example is provided to outline a proposed methodwhereby the identified region provided on a chromatography sheet withthe described luminescent agent, particularly a luminescent bacteria maybe analyzed to ascertain the chemical identity of an isolated componentsubstance of a sample.

A volume of sample containing sufficient concentration of toxicantswould be applied to a chromatography paper, such as Whatman 1M or 3M andchromatographed using a solvent system which provides maximum separationof the sample components. Various solvent systems may be utilized andtested for separation efficiency as well understood by those skilled inthe art. Small amounts of sample may be used to test for improvedresolution in one dimensional (1D) chromatography solvent systems. Thosesolvents found most effective may then be utilized for larger scaleseparation on large sheets of chromatography paper for two-dimensionalchromatography (2D).

Two dimensional chromatography may be necessary to resolve sampleingredients for subsequent identification of substantially purecompounds. By determining a combination of two solvent systems whicheffectively resolve the component toxicants, 2D chromatography can berun in duplicate.

Following the chromatography, the luminescent bacteria may be sprayedonto one of two identical sample sheets. Areas on the sheet whichdemonstrate a decreased luminescence would then be used to mark thecorresponding areas of the unsprayed sheet. The corresponding areas onthe unsprayed sheet are cut out and eluted with distilled water,appropriate solvents such as acetone or ethanol or a solvent mixture toprovide individual, substantially pure toxicants for identification.This procedure can be repeated, and/or multiple 2D sheets may be runsimultaneously, in order to accumulate sufficient quantities of varioussubstantially pure toxicants.

In this manner, appropriate amounts of toxicants in a sample may beseparated and then identified using standard chemical procedures. Forexample, small amounts of the purified component substances may be runon high pressure liquid chromatography (HPLC) and compared to knownstandards for identification¹⁵. As will be appreciated by those skilledin the art, additional standard techniques used for chemicalidentification may be employed such as spectral analysis: Mass spectra,infrared spectra (IR), nuclear magnetic resonance (NMR), and the like.

It will understood by those skilled in the art that multiple 2Dchromatography sheets can be run simultaneously in which differentsheets are sprayed with different luminescent bacteria. This wouldprovide a more thorough analysis of toxicants which may be detectable byone luminescent bacterium, but not by another. Additionally,combinations of different luminescent bacteria in one spray solution mayfacilitate the thorough identification of most or all of the detectableisolated component substances in a sample. In this manner, a thoroughanalysis and identification of toxicants in a sample may be undertaken.

Essentially this same approach can be taken using thin layerchromatography (TLC), instead of paper chromatography as describedabove, for the initial separation and identification of toxic substancesin a sample. Multiple TLC plates (e.g., Whatman 4856-840 with 1,000 μMsilica layer) may be run simultaneously in the same solvent systemutilizing 1D or 2D runs, as described above, for paper chromatography.In such a TLC approach to toxicant identification, toxicants would beidentified by spaying the plates with luminescent bacteria, marking thezones of decreased luminescence, and scraping off the correspondingareas on the unsprayed portions of plates. The scrapings are then elutedwith an appropriate solvent, such as distilled water, acetone or ethanolor a solvent mixture, concentrated (if required), and identified usingHPLC, MS, IR, NMR, and the like.

By following either of the above procedures, the separation andidentification of toxicants in a sample can be accomplished simply andrapidly. The standardization of this method to be used for theidentification of toxicants in certain types of samples will beappreciated by those skilled in the art as providing simple, rapid, andinexpensive methodologies for toxicant identification. For example,certain types of samples (i.e., industrial effluent) could be tested todetermine the initial separation system, the solvent systems, theluminescent bacteria (or combinations of luminescent bacteria), theelution protocol, and any subsequent techniques for quantitation and/oridentification.

Through the use of standard curves of easily quantitated knowncompounds, the percent recovery in a given separation system can bedetermined. In this manner, amounts of identified toxicants can bequantitated and extrapolated back to the original sample volume applied.For example, the use of radiolabeled compounds, of known specificactivity, which are separated by paper or TL chromatography, eluted, andcounted for radioactivity, would provide an indication of the percentagerecovery of a given compound. By comparing various radiolabeled chemicalcompounds in a given identification system (paper or TLC with differentsolvents and the like), one could correct for recovery losses of a givenidentification system.

When the separated toxicants are quantitated by certain chemical andspectral methods, the quantities may then be extrapolated to determinethe quantities of individual toxicants present in the original sample.Thus, this method, in many cases, would allow for toxicantquantification.

These steps could be standardized into kits tailored for the analysis ofspecific types of samples (i.e., a kit for a certain industrial effluentor certain biological samples, such as foodstuffs, pharmaceuticals, andthe like). These kits would comprise certain solvents and luminescentbacteria which would effectively resolve specific sample types therebygreatly simplifying and reducing the cost of toxicant detection,identification, and quantitation.

Alternatively, an unknown sample may be processed by the above procedurefor identification and quantitation.

Prophetic Example 10 Identification of Toxicant in a Gaseous PhaseSample with Luminescent Bacteria

The present prophetic example is provided to outline a proposed methodwhereby an investigator may identify a toxicant present in a gaseousphase sample employing the methods with luminescent bacteria describedherein.

As an initial step, the gaseous sample would be collected by techniquesknown to those skilled in the art. For example, a gas sample might becollected by filtration through a solid filter such that toxicantsdeposit onto the filter or by aspiration into a liquid such thattoxicants dissolve in the liquid. In the case of a solid filter, thefilter could then be eluted with distilled water or a suitable solvent,concentrated, chromatographed by paper or thin layer chromatography, andidentified using certain luminescent bacteria as described in Example 6.

Prophetic Example 11 Identification of a Toxicant on a Solid SurfaceSample with Luminescent Bacteria

The present prophetic example is provided to outline a proposed methodwhereby a toxicant on a solid surface sample may be identified with thedescribed luminescent bacteria.

As in Example 7, methods for removing a toxicant from a solid surface sothat it is collected in a concentrated liquid form will vary dependingon the nature of the solid surface. Techniques for such removal will beapparent to those skilled in the art. Using the procedures outlined inExample 6, one skilled in the art would be capable of identifying andquantifying toxicants which were eluted from or removed from the solidsurface. Alternatively, for direct detection of toxicants, the solidsurface could be sprayed with a certain luminescent bacteria, or mixtureof more than one luminescent bacteria, such as Vibrio fischeri and thesurface observed for zones of decreased luminescence (i.e., zones ofluminescent inhibition) substantially as has already been outlined inExample 1. Of course, these isolated component substances of the sample(potential toxicants) could then be chemically analyzed according tolaboratory techniques well known to those of skill in the art toidentify the chemical structure of the isolated component. By way ofexample, such laboratory techniques for determining the chemicalstructure of an isolated component substance include HPLC, MS, IR, NMR,and the like.

Prophetic Example 12 Proposed Test Kits for Identifying Toxicants in aSample

The present prophetic example is provided to define those componentswhich would comprise a proposed test kit useful for the identificationof toxicants in a sample. Such a kit most preferably would comprise acarrier means adapted to receive at least two container means and atleast one chromatography paper sheet in close confinement therewith. Thekit should also include at least one chromatography paper sheet and afirst container means comprising a luminescent bacterial agent. Whileany luminescent bacterial agent may be used in conjunction with thedescribed kit, that bacterial agent most preferred is the Vibriofischeri (ATTC Acc. No. 7744). Most preferably, the luminescentbacterial agent should be in lyophilized form in the container means.The lyophilized bacteria would then be suspended in a diluent solution.For example, where appropriate NaCl concentrations are within thelyophilized sample, deionized water may be employed as the diluentsolution without any expected deleterious effects to the luminescence ofthe bacteria.

In a second container means, the kit should further comprise a diluentfor a luminescent bacterial agent. Most preferably, the diluent shouldcomprise a 0.5 M NaCl buffered saline solution at pH 7 where thebacteria is a marine bacteria and has not been lyophilized to includeNaCl. The kit may optionally also include a separation solvent, such asacetonitrile, deionized water, or aqueous ammonia.

In other proposed forms of the presently proposed kit, the kit mayfurther comprise an aspirator spray bottle to facilitate the easyapplication of suspended luminescent bacteria to a separation phasematrix such as a TLC plate or chromatography paper, chromatogram. Inaddition, the kit may comprise several vials of lyophilized luminescentbacteria. In other proposed forms of the presently proposed kits, thekit may further comprise instructions for the suspension and applicationof the luminescent bacteria to facilitate visualization of the isolatedcomponent substances of the test sample, and also in regard to thereaction time to be allowed and at what point the luminescentbacteria-exposed separation phase matrix should be read.

BIBLIOGRAPHY

The following references are specifically incorporated herein byreference in pertinent part.

1. Drucker et al. (1984) E.P. 153366.

2. Vasseur et al. (1983), presented at the International Symposium onEcotoxicological Testing for Marine Environment, Belgium, pp. 12-14.

3. Baher (1988)--WPI 88-308491 (884).

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8. Ugarova et al. (1987), Appl. Biochem. Biotechnical., 15(1):35-51.

9. Thin Layer Chromatography: A Laboratory Handbook 2nd ed, E. Stahl,Ed., Springer-Varlag, New York, N.Y., (1967).

10. HPLC of Small Molecules: A Practical Approach C. K. Lim; Ed., IRLPress, Oxford England (1986).

11. HPLC of Macromolecules: A Practical Approach R. W. A. Oliver, Ed.,IRL Press, Oxford, England (1989).

12. Plant Drug Analysis: A Thin-Layer Chromatography Atlas, H. Wagner,S. Bladt, E. M. Zgainski, Springer-Verlag (eds.), New York, N.Y. (1984).

13. Alltech Bulletin, (1991) #183, Gas Chromatography Apparatus, p.11.

14. Thompson, B. C., Kugmack, J. M., Law, D. w., Winslow, J. J., eds.(1989), "Copolymeric Solid Phase Extraction for Quantitating drugs ofAbuse in Urine by Wide-Bore Capillary Gas Chromatography" L C-G-C7(10):846-850.

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19. Williamson, K. L., (1989), Macroscale and Microscale OrganicExperiments, D. C. Heath and Company, Lexington, Mass. ISBN0-669-19429-8.

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28. Armstrong, D. W., 91984) J. Liquid Chromatography, 7:353-376.

29. Rainin Scientific Catalog (1991-1992), p. 3-38.

30. Bioluminescence and Chemiluminescence: Basic Chemistry andAnalytical Applications, Marlene A. DeLuca and William D. McElroy, eds.,Academic Press (1981)

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What is claimed is:
 1. A method for identifying the presence of a toxicsubstance in a sample using a luminescent biological agent said methodcomprising the steps of:preparing a luminescent biological agentsuitable for use in conjunction with a separation phase matrix;obtaining a sufficient volume of the sample to comprise a test samplesuspected to contain toxic substances; separating the toxic substancespresent in the test sample by applying the test sample to a separationphase matrix to provide separated toxic substances of the test sample;and exposing the separation phase matrix to the luminescent biologicalagent, identifying the presence of a toxic substance by detecting regionof inhibition of luminescence on the separation phase matrix.
 2. Themethod of claim 1 wherein the luminescent biological agent is selectedfrom the group consisting of a luminescent bacteria, a luminescentfungi, a luminescent fish extract, a luminescent dinoflagellate, aluminescent firefly extract, a luminescent anthrozoan, a luminescentearthworm extract, a luminescent coelenterate extract, and a luminescentcrustacean.
 3. The method of claim 1 wherein the luminescent biologicalagent is a luminescent bacteria.
 4. The method of claim 3 where theluminescent bacteria is selected from the group consisting ofPhotobacterium leiognathi, Photobacterium phosphoreum, Vibrio fischeri(ATCC Acc. 7744) or Vibrio harveyi (ATCC Acc. No. 33843).
 5. The methodof claim 1 wherein the luminescent biological agent is Vibrio fischeri,(ATCC Acc. No. 7744) or Vibrio harveyi (ATCC Acc. No. 33843).
 6. Themethod of claim 1 wherein the luminescent biological agent is Vibriofischeri (ATCC Acc. No. 7744).
 7. The method of claim 1 wherein the testsample is a liquid sample, a solid sample or a gaseous sample.
 8. Themethod of claim 7 wherein the separation phase matrix is chromatographypaper and the test sample is a liquid sample.
 9. The method of claim 8wherein the separation phase matrix is exposed to a luminescentbacterium in a suspension suitable for spraying.
 10. The method of claim9 wherein the presence of separated toxic substances of the test sampleis indicated by a zone of bacterial luminescent inhibition on theseparation phase matrix.
 11. The method of claim 1 wherein the testsample comprises garlic, pesticides, herbicides or heavy metals.
 12. Themethod of claim 9 wherein a volume of the luminescent bacteria comprisesa suspension of 10⁸ -10⁹ bacterial cells/ml.
 13. A method foridentifying the presence of a toxic substance in a sample using aluminescent biological agent said method comprising the stepsof:preparing a luminescent biological agent which is inhibited by asubstance which is toxic to an organism; obtaining a sufficient volumeof the sample suspected to contain toxic substances which are toxic toan organism to provide a test sample; separating the toxic substancespresent in the test sample on a separation phase matrix to provideseparated toxic substances; and exposing the separation phase matrix toa concentration of the luminescent biological agent effective to formzones of luminescent inhibition; and identifying the presence of saidtoxic substance harmful to an organism in the sample, by zones ofluminescent inhibition on the separation phase matrix.
 14. A method forchemically identifying a toxic substance in a sample using a luminescentbacterial or luminescent firefly extract said method comprising thesteps of:preparing a luminescent bacterial or luminescent fireflyextract suitable for use in conjunction with a separation phase matrix;obtaining a sufficient volume of the sample suspected to contain toxicsubstances to provide a test sample; separating the toxic substances ofthe test sample on a separation phase matrix to provide a first sampleseparation phase matrix; exposing the first sample separation phasematrix to the luminescent bacterial or luminescent firefly extract;identifying the presence of said toxic substances in the sample, saidpresence being indicated by zones of luminescent inhibition on the firstsample separation phase matrix; obtaining a second volume of the sampleto form a second test sample; separating the toxic substances of thesecond test sample on another separation phase matrix to provide asecond separation phase matrix; determining the chemical identity of aseparated toxic substance observed at areas of luminescent inhibition onthe first sample separation phase matrix by analyzing a correspondinginhibition region on the second separation phase matrix.
 15. The methodof claim 13 wherein the toxic substances are harmful to a virus.
 16. Themethod of claim 13 wherein the organism is a plant, an animal or amicroorganism.
 17. The method of claim 16 wherein the animal is a human.18. The method of claim 13 or 14 wherein the toxic substances areselected from the group consisting of:pesticides; herbicides; heavymetals; and plant extracts.
 19. The method of claim 13 or 14 wherein thetoxic substance is a pesticide selected from the group consisting ofO,O,diethyl-O-(2-isopropyl-6-methyl-4-pyrmidinyl) phosphorothionate,gamma isomer of benzene hexachloride liquid and 1-naphthyl-N-methylcarbamate.
 20. The method of claim 13 or 14 wherein the toxic substanceis a herbicide selected from the group consisting of isopropylamino saltof glycophosphate-n-(phosphoromethyl) glycine or a mixture ofdimethylamino salt of 2,4 dichlorophenoxyacetic acid and dimethylaminosalt of 2-(2-methyl-4 chlorophenoxy) propionic acid.
 21. The method ofclaim 13 or 14 wherein the toxic substance is a heavy metal selectedfrom the group consisting of mercury, lead or cadmium, and salt thereof.22. The method of claim 13 wherein the luminescent biological agent is aluminescent bacteria.
 23. The method of claim 13 or 14 wherein theluminescent biological agent is selected from the group consisting ofPhotobacterium phosphoreum, Vibrio fischeri, (ATCC Acc. No. 7744) Vibrioharveyi (ATCC Acc. No. 33843) and Photobacterium leiognathi.
 24. Themethod of claim 13 or 14 wherein the luminescent biological agent isselected from the group consisting of Vibrio fischeri (ATCC Acc. No.7744) and Vibrio harveyi (ATCC Acc. No. 33843).
 25. The method of claim13 wherein the luminescent biological agent is a suspension ofluminescent bacteria containing about 10⁸ -10⁹ bacteria cells/ml. 26.The method of claim 14 wherein the chemical identity of the separatedtoxic substance of the test sample is achieved by analyzing thecorresponding inhibition region of the sample on the second separationphase matrix by HPLC, nuclear mass spectrometry, infrared spectroscopy,mass spectroscopy or electron capture detection.
 27. The method of claim13 or 14 wherein the separation phase matrix is a thin layerchromatography plate and wherein the separated toxic substances areexposed to a suspension of luminescent bacteria.
 28. The method of claim25 wherein the luminescent bacteria is sprayed onto the thin layerchromatography plate to provide zones of inhibition to identify thepresence of the toxic substance in the test sample.
 29. The method ofclaim 25 wherein the luminescence of the bacterial agent is notinhibited by Volck oil spray or calcium ion.
 30. The method of claim 25wherein the inhibition of luminescence of the bacterial agent is greaterfor a toxic substance comprisingO,O,diethyl-O-(2-isopropyl-6-methyl-4-pyrmidinyl)phosphorothio(n)atethan for a toxic substance comprising gamma isomer of benzenehexachloride liquid.
 31. The method of claim 25 wherein a solvent isused to separate toxic substances of the sample on the thin layerchromatograph sheet, and the solvent is selected from the groupconsisting of ETOH, Hexane/THF and acetonitrile/water/aqueous ammonia.32. A kit for the identification of the presence of a toxic in a sampleusing a luminescent biological agent, said kit comprising:a carriermeans adapted to receive at least two container means and at least oneseparation phase matrix in close confinement therewith; at least oneseparation phase matrix, wherein said separation phase matrix is a thinlayer chromatography plate; a first container means comprising aluminescent biological agent; and a second container means comprising adiluent for the luminescent biological agent.
 33. The kit of claim 32wherein the luminescent biological agent is a luminescent bacteriaselected from the group consisting of Photobacterium phosphoreum, Vibriofischeri (ATCC Acc. No. 7744), Vibrio harveyi (ATCC No. 33843) orPhotobacterium leiognathi.
 34. The kit of claim 32 wherein theluminescent biological agent is a luminescent bacteria selected from thegroup consisting of Vibrio fischeri (ATCC No.7744) and Vibrio harveyi(ATCC 33843).
 35. The kit of claim 32 wherein the luminescent biologicalagent is a luminescent bacteria, Vibrio fischeri (ATCC Acc. No. 7744).36. The kit of claim 32 wherein the luminescent biological agent is abacterial agent in a lyophilized form.
 37. The kit of claim 32 whereinthe diluent is a saline solution comprising between 1%-3% NaCl wt/vol.38. The kit of claim 32 wherein the diluent is an about 0.5 M NaClsaline solution.
 39. A method for identifying the presence of abiologically toxic substances in a sample using a luminescent bacteriaselected from the group consisting of Vibrio fischeri, Photobacteriumleiognathi, Vibrio harveyi, or Photobacterium phosphoreumcomprising:preparing the Vibrio fischeri, Photobacterium leiognathi,Vibrio harveyi, or Photobacterium phosphoreum luminescent reagent;separating the toxic substances in the test sample into separated zoneson a first thin layer chromatography sheet; exposing the first thinlayer chromatography sheet to a biological toxin indicating amount ofthe luminescent reagent and observing zones of luminescence and zones ofluminescent inhibition on the thin layer chromatography sheet; obtaininga second volume of the sample and separating the toxic substances of thesecond test sample on a second thin layer chromatography sheet; andidentifying the presence of the separated toxic substances of the sampleat areas of luminescent inhibition on the exposed thin layerchromatography sheet by analyzing the corresponding zone of inhibitionof the toxic substance on the second thin layer chromatography sheet.40. The method of claim 39 wherein the toxic substance is selected fromthe group consisting of copper, lead, mercury,O,O,diethyl-O-(2-isopropyl-6-methyl-4-pyrmidinyl)phosphorothio(n)ate,gamma isomer of benzene hexachloride liquid, 1-naphthyl-N-methylcarbamate, a mixture of dimethylamino salt of 2,4 dichlorophenoxyaceticacid and dimethylamino salt of 2-(2-methyl-4 chlorophenoxy) propionicacid, isopropylamino salt of glycophosphate N-(phosphoromethyl)glycine,nicotine sulfates, Volk Oil spray, and cyanide.
 41. The method of claim39 wherein the luminescent-bacterial agent is Vibrio fischeri orPhotobacterium leiognathi.